T Cell Modification and Use Thereof

ABSTRACT

This invention relates to modified T cells that inducibly express a bioactive molecule, such as IL-7, and constitutively expresses an antigen receptor, such as a T cell receptor or chimeric antigen receptor that binds to a tumour antigen. The modified T cells may comprise a nucleic acid construct that comprises a first nucleotide sequence encoding the bioactive molecule, a second nucleotide sequence encoding the antigen receptor; an inducible promoter operably linked to the first nucleotide sequence and a constitutive promoter operably linked to the second nucleotide. Nucleic acid constructs and vectors are provided, as well as T cells comprising such constructs and vectors and therapeutic methods and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of United KingdomApplication No. GB 1713078.2 filed Aug. 15, 2017, the disclosure ofwhich is incorporated herein by reference in its entirety.

FIELD

The present invention relates to the modification of T cells to increasetheir cytotoxic activity and the use of modified T cells inimmunotherapy, for example for the treatment of cancer.

BACKGROUND

T cells (or T lymphocytes) are found widely distributed within tissuesand the tumour environment. T cells are distinguished from otherlymphocytes by the presence of T cell receptors (TCRs) on the cellsurface. The TCR is a multi-subunit transmembrane complex that mediatesthe antigen-specific activation of T cells. The TCR confers antigenspecificity on the T cell, by recognising an antigen peptide ligand thatis presented on the target cell by a major histocompatibility complex(MHC) molecule.

Although peptides derived from altered or mutated proteins in tumourtarget cells can be recognised as foreign by T cells expressing specificTCRs, many antigens on tumour cells are simply upregulated oroverexpressed (so called self-antigens) and do not induce a functional Tcell response. Therefore, studies have focussed on identifying targettumour antigens which are expressed, or highly expressed, in themalignant but not the normal cell type. Examples of such targets includethe cancer/testis (CT) antigen NY-ESO-1, which is expressed in a widearray of human cancers but shows restricted expression in normal tissues(Chen Y-T et al. Proc Natl Acad Sci USA. 1997; 94(5):1914-1918), and theMAGE-A family of CT antigens which are expressed in a very limitednumber of healthy tissues (Scanlan M. J. et al. Immunol Rev. 2002;188:22-32).

Identification of such antigens has promoted the development of targetedT cell-based immunotherapy, which has the potential to provide specificand effective cancer therapy (Ho, W. Y. et al. Cancer Cell 2003;3:1318-1328; Morris, E. C. et al. Clin. Exp. Immunol. 2003; 131:1-7;Rosenberg, S. A. Nature 2001; 411:380-384; Boon, T. and van der BruggenP. J. Exp. Med. 1996; 183:725-729).

The intravenous administration of interleukin 7 (IL-7) has been proposedto improve outcomes in T cell-based immunotherapy. IL-7 is known tobolster the persistence of tumour-specific T-cells (Melchionda, F. etal. J. Clin. Invest. 2005; 115:1177-87), and T-cells geneticallymodified to either secrete IL-7 or overexpress the IL-7 receptor (inconjunction with administered IL-7) display enhanced antitumour efficacyin preclinical models (Vera, J. F. et al. Mol. Ther. 2009; 17:880-8,Markley, J. C. and Sadelain, M. Blood 2010; 115:3508-3519). However,systemic administration of cytokines to patients with cancer has causedsignificant toxicity (Sportes, C. et al. Clin. Cancer Res. 2010;16:727-35, Conlon, K. C. t al. J. Clin. Oncol. 2015; 33:74-82, Brudno,J. N. et al. Blood 2016; 127:3321-31) Alternative approaches such asgenetic modification of T-cells to secrete or trans-present cytokines(Hutton L. V. et al. Proc. Natl. Acad. Sci. USA 2016; 113:E7788-97)carry a risk of severe adverse events, including neurotoxicity andcytokine release syndrome from systemic accumulation of secretedcytokine (Zhang, L. et al. Clin. Cancer Res. 21; 21:2278-88), whereasT-cells that overexpress cytokine receptors do not eliminate the needfor exogenous cytokine (Vera, J. F. et al. Mol. Ther. 2009; 17:880-8).Therefore a method for safely delivering cytokines, such as IL-7, andother bioactive molecules, to T-cells remains elusive.

SUMMARY

The present inventors have unexpectedly recognised that T cellscontaining a nucleic acid construct that provides constitutiveexpression of an antigen receptor and inducible expression of abioactive molecule, such as Interleukin 7 (IL-7), upon T cellactivation, display improved anti-tumour properties without the hightoxicity that is observed when the bioactive molecule is expressedconstitutively in T cells.

A first aspect of the invention provides a nucleic acid constructcomprising;

-   -   (i) a first nucleotide sequence encoding a bioactive molecule;    -   (ii) a second nucleotide sequence encoding an antigen receptor;    -   (iii) an inducible promoter operably linked to the first        nucleotide sequence and    -   (iv) a constitutive promoter operably linked to the second        nucleotide.

Preferably the bioactive molecule is a cytokine, most preferably IL-7.

A second aspect of the invention provides a vector, for example alentiviral vector, comprising a nucleic construct of the first aspect.

A third aspect of the invention provides a population of T cellscomprising a nucleic construct or a vector according to the first orsecond aspect.

A fourth aspect of the invention provides a population of T cells whichconstitutively express a heterologous antigen receptor and induciblyexpress a bioactive molecule, such as IL-7, upon T cell activation.

A fifth aspect of the invention provides a pharmaceutical compositioncomprising a population of T cells according to the third or fourthaspects and a pharmaceutically acceptable excipient.

A fifth aspect of the invention provides a population of T cellsaccording to the third or fourth aspects for use in a method oftreatment of the human or animal body, for example a method of treatmentof cancer in an individual. Related aspects provide the use of apopulation of T cells according to the third or fourth aspects in themanufacture of a medicament for the treatment of cancer in an individualand a method of treating cancer comprising administering to anindividual with cancer a population of T cells according to the third orfourth aspects.

A sixth aspect of the invention provides a method of producing apopulation of modified T cells comprising;

-   -   introducing an nucleic acid construct or a vector according to        the first or second aspects into a population of T cells        obtained from a donor individual to produce a population of        modified T cells.

A seventh aspect of the invention provides a method of treating cancerin an individual in need thereof comprising;

-   -   introducing an nucleic acid construct or a vector according to        the first or second aspects into a population of T cells        obtained from a donor individual to produce a population of        modified T cells, and    -   administering the population of modified T cells to a recipient        individual.

The donor individual and the recipient individual may be the same (i.e.autologous treatment; the modified T cells are obtained from anindividual who is subsequently treated with the modified T cells) or thedonor individual and the recipient individual may be different (i.e.allogeneic treatment; the modified T cells are obtained from oneindividual and subsequently used to treat a different individual).

Other aspects and embodiments of the invention are described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sequence and annotation of the NFAT inducible IL-7expression insert. The sequence illustrated in a 5′ to 3′ orientationwith individual components of the cassette highlighted. These includethree copies of the NFAT IL-2 TRE GGAGGAAAAACTGTTTCATACAGAAGGCGT,minimal CMV promoter, IL-7 coding sequence (codon optimised) and theSV40 polyA.

FIG. 2 shows a map of the NFAT-IL-7 plasmid supplied by GeneArt. TheNFAT-IL-7 cassette was removed by digestion with AgeI and MluI.

FIG. 3 shows a vector map of the lentiviral vector expressing the TCR1.Highlighted are the unique AgeI and MluI restriction

FIG. 4 shows a vector map of construct ADB967. The NFAT-IL-7 cassettewas cloned between the AgeI and MluI restriction sites. Transcription ofIL-7 occurs from the antisense strand and in a reverse orientation tothat of the TCR1.

FIG. 5 shows a vector map of ADB1099 IL7_T2A_TCR1. IL7_T2A_TCR1 isexpressed as a single ORF with IL-7, TCRα and TCRβ chains separated by2A like skip sequences.

FIG. 6 shows a plasmid map for NFAT inducible IL-7 TCR2

FIG. 7 shows a vector map of constitutive IL-7_(—) TCR2. The IL-7_TCR2was cloned into the vector AB1224 between the NheI and SalI sites.

FIG. 8 shows a schematic of the design of the three main lentiviralconstructs used in this study. Constructs were produced that expressIL-7 under the control of 3 repeats of the NFAT promotor derived fromthe IL-2 promotor sequence in one direction or IL-7 constitutively inthe presence SPEAR TCR and constitutive expression of the SPEAR TCR.Note: the sizes of the various components of the constructs are notshown to-scale.

FIG. 9 shows an assessment of the percentage of CD4+ and CD8+ T cellsand of T cell transduction efficiency. NTD T cells (white circles), Tcells transduced with TCR (black squares), constitutive IL-7_TCR (blacktriangles) or with NFAT_IL-7_TCR (black circles). Data shows mean+/−SD.(A) The data are shown as the percentage of CD3+ T cells that wereeither CD4+ or CD8+. (B) The data are shown as the percentage of cellsthat were CD3+ and either CD4+ or CD8+ and that were TCR Vα24+. (C) Thedata are shown as the mean fluorescence intensity (MFI) in either CD4+or CD8+ Vα24+ population.

FIG. 10 shows an assessment of 4-1BB, CD127, CD27, CD28, CD40L, OX-40,PD-1 and TIM-3 expression on CD4+ T cells. NTD T cells (white circles),T cells transduced with TCR (black squares), constitutive IL-7_TCR(black triangles) or with NFAT_IL-7_TCR (black circles). Data showsmean+/−SD. The data are shown as the percentage of cells were either4-1BB+, CD127+, CD27+, CD28+, CD40L+, OX-40+, PD-1+ or TIM-3+ and either(A) CD4+ (B) CD4+ Vα24+ or (B) CD4+ Vα24.

FIG. 11 shows an assessment of 4-1BB, CD127, CD27, CD28, CD40L, OX-40,PD-1 and TIM-3 expression on CD8+ T cells. NTD T cells (white circles),T cells transduced with MAGE-A4c1032 TCR (black squares), constitutiveIL-7_MAGE-A4c1032 TCR (black triangles) or withNFAT_IL-7_MAGE-A4c1032TCR (black circles). Data shows mean+/−SD. Thedata are shown as the percentage of cells were either 41BB+, CD127+,CD27+, CD28+, CD40L+, OX-40+, PD-1+ or TIM-3+ and either (A) CD8+ (B)CD8+ Vα24+ or (B) CD8+ Vα24−

FIG. 12 shows an assessment of the proportions of naïve and memory CD4+T cells. NTD T cells (white circles), T cells transduced with TCR (blacksquares), constitutive IL-7_TCR (black triangles) or with NFAT_IL-7_TCR(black circles). Data shows mean+/−SD. The data are shown as thepercentage of cells were either CCR7− CD45RA− CD45RO−, CCR7− CD45RA−CD45RO++, CCR7− CD45RA+ CD45RO−, CCR7− CD45RA+ CD45RO+, CCR7+ CD45RA−CD45RO−, CCR7+ CD45RA− CD45RO+, CCR7+ CD45RA+ CD45RO− or CCR7+ CD45RA+CD45RO+ and either (A) CD4+ (B) CD4+Vα24+ or (B) CD4+ Vα24−.

FIG. 13 shows an assessment of the proportions of naïve and memory CD8+T cells. NTD T cells (white circles), T cells transduced with TCR (blacksquares), constitutive IL-7_TCR (black triangles) or with NFAT_IL-7_TCR(black circles). Data shows mean+/−SD. The data are shown as thepercentage of cells were either CCR7− CD45RA− CD45RO−, CCR7− CD45RA−CD45RO+, CCR7− CD45RA+ CD45RO−, CCR7− CD45RA+ CD45RO+, CCR7+ CD45RA−CD45RO−, CCR7+ CD45RA− CD45RO+, CCR7+ CD45RA+ CD45RO− or CCR7+ CD45RA+CD45RO+ and either (A) CD8+ (B) CD8+ Vα24+ or (B) CD8+ Vα24−.

FIG. 14 shows IL-7 production by T cells in response to antigen positiveand negative cells. NTD T cells (white circles), T cells transduced withTCR (black squares), constitutive IL-7_TCR (black triangles) or withNFAT_IL-7_TCR (black circles) were incubated 48 hours alone, or withA375, Mel624 or Colo205 cells. A375 cells pulsed with exogenous peptideor CD3/CD28 beads were included as controls. Data were from 6 donors.Supernatants from T cells were either diluted 1:5 of 1:10. The maximumconcentration on the standard curve was 1000 pg/ml and as such anyvalues >2000 pg/ml, 5000 pg/ml or 10.000 pg/ml were assigned a value of2000 pg/ml, 5000 pg/ml or 10.000 pg/ml respectively. Data showsmean+/−SD of triplicate wells.

FIG. 15 shows total T cell counts from donors FXA-006 and FFA-004 inresponse to repeated stimulation with irradiated A375 cells (Assay ref.no.: ACF1141). Total live cell counts (Trypan Blue negative cells) fromco-cultures with NTD T cells (white circles), T cells transduced withTCR (black squares), constitutive IL-7_TCR (black triangles) or withNFAT_IL-7_TCR (black circles) that were restimulated weekly withirradiated A375 (HLA-A2+/NY-ESO+) cells in the presence or absence of 20ng/ml IL-7. Restimulation was performed on days 0, 7, 14 and 21 and isindicated by the arrows. T cells were from donors (A) FXA-006 or (B)FFA-004 in the absence of IL-7 or from (C) FXA-006 or (D) FFA-004 in thepresence of IL-7. The total cell count was assessed on days 7, 14, 21and 28.

FIG. 16 shows that the survival of mice injected with T cellsco-expressing inducible IL-7 with an engineered TCR compared with Tcells expressing TCR alone or TCR together with constitutive IL-7.Survival curves are shown for mice injected i.v. with 1×10⁶ Mel624tumour cells on day 0, followed by i.v. injection of 2×10⁶ total T cellson day 7 as indicated. The percentage of transduced T cells (TCRVβ+/CD3+) were normalised to ˜42% prior to injection using NTD T cells.Mice were culled as poor condition/weight loss required and the graphsindicate the percent survival of each group over the time of the study.(A-E) Percentage survival of mice injected with tumour alone (solidline) or the various T cells as indicated (dashed and dotted lines).

DETAILED DESCRIPTION

This invention relates to modified T cells that inducibly express abioactive molecule, such as IL-7, and constitutively expresses anantigen receptor. The modified T cells may comprise a nucleic acidconstruct that comprises;

-   -   (i) a first nucleotide sequence encoding a bioactive molecule    -   (ii) a second nucleotide sequence encoding an antigen receptor;    -   (iii) an inducible promoter operably linked to the first        nucleotide sequence and    -   (iv) a constitutive promoter operably linked to the second        nucleotide.

T cells (also called T lymphocytes) are white blood cells that play acentral role in cell-mediated immunity. T cells can be distinguishedfrom other lymphocytes by the presence of a T cell receptor (TCR) on thecell surface. There are several types of T cells, each type having adistinct function.

T helper cells (T_(H) cells) are known as CD4⁺ T cells because theyexpress the CD4 surface glycoprotein. CD4⁺ T cells play an importantrole in the adaptive immune system and help the activity of other immunecells by releasing T cell cytokines and helping to suppress or regulateimmune responses. They are essential for the activation and growth ofcytotoxic T cells.

Cytotoxic T cells (Tc cells, CTLs, killer T cells) are known as CD8⁺ Tcells because they express the CD8 surface glycoprotein. CD8⁺ T cellsact to destroy virus-infected cells and tumour cells. Most CD8⁺ T cellsexpress TCRs that can recognise a specific antigen displayed on thesurface of infected or damaged cells by a class I MHC molecule. Specificbinding of the TCR and CD8 glycoprotein to the antigen and MHC moleculeleads to T cell-mediated destruction of the infected or damaged cells.

T cells for use as described herein may be CD4⁺ T cells; CD8⁺ T cells;or CD4⁺ T cells and CD8⁺ T cells. For example, the T cells may be amixed population of CD4⁺ T cells and CD8⁺ T cells.

Suitable T cells for use as described herein may be obtained from adonor individual. In some embodiments, the donor individual may be thesame person as the recipient individual to whom the T cells will beadministered following modification and expansion as described herein(autologous treatment). In other embodiments, the donor individual maybe a different person to the recipient individual to whom the T cellswill be administered following modification and expansion as describedherein (allogeneic treatment). For example, the donor individual may bea healthy individual who is human leukocyte antigen (HLA) matched(either before or after donation) with a recipient individual sufferingfrom cancer.

A method described herein may comprise the step of obtaining T cellsfrom a donor individual and/or isolating T cells from a sample obtainedfrom a donor individual with cancer.

A population of T cells may be isolated from a blood sample. Suitablemethods for the isolation of T cells are well known in the art andinclude, for example fluorescent activated cell sorting (FACS: see forexample, Rheinherz et al (1979) PNAS 76 4061), cell panning (see forexample, Lum et al (1982) Cell Immunol 72 122) and isolation usingantibody coated magnetic beads (see, for example, Gaudernack et al 1986J Immunol Methods 90 179). CD4⁺ and CD8⁺ T cells may be isolated fromthe population of peripheral blood mononuclear cells (PBMCs) obtainedfrom a blood sample. PBMCs may be extracted from a blood sample usingstandard techniques. For example, ficoll may be used in combination withgradient centrifugation (Böyum A. Scand J Clin Lab Invest. 1968;21(Suppl. 97):77-89), to separate whole blood into a top layer ofplasma, followed by a layer of PBMCs and a bottom fraction ofpolymorphonuclear cells and erythrocytes. In some embodiments, the PBMCsmay be depleted of CD14⁺ cells (monocytes).

Following isolation, the T cells may be activated. Suitable methods foractivating T cells are well known in the art. For example, the isolatedT cells may be exposed to a T cell receptor (TCR) agonist. Suitable TCRagonists include ligands, such as a peptide displayed on a class I or IIMHC molecule on the surface of an antigen presenting cell, such as adendritic cell, and soluble factors, such as anti-TCR antibodies.

An anti-TCR antibody may specifically bind to a component of the TCR,such as εCD3, αCD3 or αCD28. Anti-TCR antibodies suitable for TCRstimulation are well-known in the art (e.g. OKT3) and available fromcommercial suppliers (e.g. eBioscience CO USA). In some embodiments, Tcells may be activated by exposure to anti-αCD3 antibodies and IL2. Morepreferably, T cells are activated by exposure to anti-αCD3 antibodiesand anti-αCD28 antibodies. The activation may occur in the presence orabsence of CD14⁺ monocytes. Preferably, the T cells may be activatedwith anti-CD3 and anti-CD28 antibody coated beads. For example, PBMCs orT cell subsets including CD4⁺ and/or CD8⁺ cells may be activated,without feeder cells (antigen presenting cells) or antigen, usingantibody coated beads, for example magnetic beads coated with anti-CD3and anti-CD28 antibodies, such as Dynabeads® Human T-Activator CD3/CD28(ThermoFisher Scientific).

Following isolation and activation, the T cells may be modified toincorporate the nucleic acid construct.

The bioactive molecule and antigen receptor expressed in the modified Tcell are recombinant proteins that are encoded by heterologous nucleicacid i.e. the bioactive molecule and the antigen receptor are expressedfrom encoding nucleic acid that has been incorporated into the T cell byrecombinant techniques.

Modification of a T cell to express the bioactive molecule and theantigen receptor may comprise introducing the nucleic acid constructinto the T cell. Suitable methods for the introduction and expression ofheterologous nucleic acids into T cells are well-known in the art anddescribed in more detail below.

The bioactive molecule may be a growth factor or a cytokine, preferablyInterleukin 7 (IL-7). Interleukin 7 (IL-7) may be human IL-7 and mayhave the amino acid sequence of SEQ ID NO: 3.

Expression of IL-7 from the inducible promoter is induced by T-cellactivation.

The inducible promoter may comprise a nuclear factor of activated Tcells (NFAT)/AP1 transcriptional response element (TRE). Uponrecognition of the cognate peptide MHC1 complex, NFAT undergoes Ca2+dependent translocation to the nucleus where it promotes transcriptionof genes which harbour an NFAT TRE. Suitable NFAT TREs are well-known inthe art and include the human IL2 promoter NFAT TRE (Macian et al (2001)Oncogene. 2001 Apr. 30; 20(19):2476-89) which has the sequence of SEQ IDNO: 14 or a variant thereof.

The inducible promoter may comprise one, two, three or more repeats ofthe NFAT TRE.

The inducible promoter may further comprise additional promoterelements, for example a minimal viral promoter such as CMV. Suitablepromoter elements are well known in the art and include the minimal CMVpromoter of SEQ ID NO: 15 or a variant thereof.

A suitable inducible promoter sequence operably linked to a nucleotidesequence encoding IL-7 may comprise the nucleotide sequence of SEQ IDNO: 1 or a variant thereof

Expression from the constitutive promoter does not vary in response totranscription factors and the second nucleic acid sequence is expressedcontinuously in the T cell. Suitable constitutive promoters are wellknown in the art and include mammalian promoters, such as Humanelongation factor-1 alpha (EF1α).

A suitable antigen receptor may bind specifically to target cells,preferably cancer cells.

The antigen receptor may be a T cell receptor (TCR). TCRs aredisulphide-linked membrane anchored heterodimeric proteins, typicallycomprising highly variable alpha (a) and beta (β) chains expressed as acomplex with invariant CD3 chain molecules. T cells expressing thesetype of TCRs are referred to as α:β (or αβ) T cells. A minority of Tcells express an alternative TCR comprising variable gamma (γ) and delta(δ) chains and are referred to as γδ T cells.

Suitable TCRs bind specifically to a major histocompatibility complex(MHC) on the surface of cancer cells that displays a peptide fragment ofa tumour antigen. An MHC is a set of cell-surface proteins which allowthe acquired immune system to recognise ‘foreign’ molecules. Proteinsare intracellularly degraded and presented on the surface of cells bythe MHC. MHCs displaying ‘foreign’ peptides, such a viral or cancerassociated peptides, are recognised by T cells with the appropriateTCRs, prompting cell destruction pathways. MHCs on the surface of cancercells may display peptide fragments of tumour antigen i.e. an antigenwhich is present on a cancer cell but not the correspondingnon-cancerous cell. T cells which recognise these peptide fragments mayexert a cytotoxic effect on the cancer cell.

Suitable TCRs are well known in the art and include the TCRs of SEQ IDNOs: 6 and 11 and variants thereof.

In some embodiments, the coding sequences for the individual chains ofthe TCR (e.g. TCRα and TCRβ chains) may be separated by a cleavagerecognition sequence. This allows the chains of the TCR to be expressedas a single fusion which undergoes intracellular cleavage to generatethe two separate proteins. Suitable cleavage recognition sequences arewell known in the art and include 2A-furin sequence.

Preferably, the TCR is not naturally expressed by the T cells (i.e. theTCR is exogenous or heterologous). Heterologous TCRs may include αβTCRheterodimers. Suitable heterologous TCRs may bind specifically to cancercells that express a tumour antigen. For example, the T cells may bemodified to express a heterologous TCR that binds specifically to MHCsdisplaying peptide fragments of a tumour antigen expressed by the cancercells in a specific cancer patient. Tumour antigens expressed by cancercells in the cancer patient may identified using standard techniques.

A heterologous TCR may be a synthetic or artificial TCR i.e. a TCR thatdoes not exist in nature. For example, a heterologous TCR may beengineered to increase its affinity or avidity for a tumour antigen(i.e. an affinity enhanced TCR). The affinity enhanced TCR may compriseone or more mutations relative to a naturally occurring TCR, forexample, one or more mutations in the hypervariable complementaritydetermining regions (CDRs) of the variable regions of the TCR α and βchains. These mutations increase the affinity of the TCR for MHCs thatdisplay a peptide fragment of a tumour antigen expressed by cancercells. Suitable methods of generated affinity enhanced TCRs includescreening libraries of TCR mutants using phage or yeast display and arewell known in the art (see for example Robbins et al J Immunol (2008)180(9):6116; San Miguel et al (2015) Cancer Cell 28 (3) 281-283; Schmittet al (2013) Blood 122 348-256; Jiang et al (2015) Cancer Discovery 5901).

Preferred affinity enhanced TCRs may bind to cancer cells expressing oneor more of the tumour antigens NY-ESO1, PRAME, alpha-fetoprotein (AFP),MAGE A4, MAGE A1, MAGE A10 and MAGE B2.

Alternatively, the antigen receptor may be a chimeric antigen receptor(CAR). CARs are artificial receptors that are engineered to contain animmunoglobulin antigen binding domain, such as a single-chain variablefragment (scFv). A CAR may, for example, comprise an scFv fused to a TCRCD3 transmembrane region and endodomain. An scFv is a fusion protein ofthe variable regions of the heavy (V_(H)) and light (V_(L)) chains ofimmunoglobulins, which may be connected with a short linker peptide ofapproximately 10 to 25 amino acids (Huston J. S. et al. Proc Natl AcadSci USA 1988; 85(16):5879-5883). The linker may be glycine-rich forflexibility, and serine or threonine rich for solubility, and mayconnect the N-terminus of the V_(H) to the C-terminus of the V_(L), orvice versa. The scFv may be preceded by a signal peptide to direct theprotein to the endoplasmic reticulum, and subsequently the T cellsurface. In the CAR, the scFv may be fused to a TCR transmembrane andendodomain. A flexible spacer may be included between the scFv and theTCR transmembrane domain to allow for variable orientation and antigenbinding. The endodomain is the functional signal-transmitting domain ofthe receptor. An endodomain of a CAR may comprise, for example,intracellular signalling domains from the CD3 ζ-chain, or from receptorssuch as CD28, 41BB, or ICOS. A CAR may comprise multiple signallingdomains, for example, but not limited to, CD3z-CD28-41BB orCD3z-CD28-OX40.

The CAR may bind specifically to a tumour-specific antigen expressed bycancer cells. For example, the T cells may be modified to express a CARthat binds specifically to a tumour antigen that is expressed by thecancer cells in a specific cancer patient. Tumour antigens expressed bycancer cells in the cancer patient may identified using standardtechniques.

Expression of a heterologous antigen receptor, such as a heterologousTCR or CAR, may alter the immunogenic specificity of the T cells so thatthey recognise or display improved recognition for one or more tumourantigens that are present on the surface of the cancer cells of anindividual with cancer.

In some embodiments, the T cells may display reduced binding or nobinding to cancer cells in the absence of the heterologous antigenreceptor. For example, expression of the heterologous antigen receptormay increase the affinity and/or specificity of the cancer cell bindingof modified T cells relative to unmodified T cells.

The term “heterologous” refers to a polypeptide or nucleic acid that isforeign to a particular biological system, such as a host cell, and isnot naturally present in that system. A heterologous polypeptide ornucleic acid may be introduced to a biological system by artificialmeans, for example using recombinant techniques. For example,heterologous nucleic acid encoding a polypeptide may be inserted into asuitable expression construct which is in turn used to transform a hostcell to produce the polypeptide. A heterologous polypeptide or nucleicacid may be synthetic or artificial or may exist in a differentbiological system, such as a different species or cell type. Anendogenous polypeptide or nucleic acid is native to a particularbiological system, such as a host cell, and is naturally present in thatsystem. A recombinant polypeptide is expressed from heterologous nucleicacid that has been introduced into a cell by artificial means, forexample using recombinant techniques. A recombinant polypeptide may beidentical to a polypeptide that is naturally present in the cell or maybe different from the polypeptides that are naturally present in thatcell.

A variant of a reference amino acid or nucleotide sequence set outherein may comprise a sequence having at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 98%sequence identity to the reference sequence. Particular amino acidsequence variants may differ from a repeat domain shown above byinsertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more than 10 amino acids. Particular nucleotidesequence variants may differ from a reference sequence set out herein byinsertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more than 10 amino acids.

Sequence similarity and identity are commonly defined with reference tothe algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAPuses the Needleman and Wunsch algorithm to align two complete sequencesthat maximizes the number of matches and minimizes the number of gaps.Generally, default parameters are used, with a gap creation penalty=12and gap extension penalty=4. Use of GAP may be preferred but otheralgorithms may be used, e.g. BLAST (which uses the method of Altschul etal. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method ofPearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Watermanalgorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or theTBLASTN program, of Altschul et al. (1990) supra, generally employingdefault parameters. In particular, the psi-Blast algorithm (Nucl. AcidsRes. (1997) 25 3389-3402) may be used.

Sequence comparison may be made over the full-length of the relevantsequence described herein.

The heterologous antigen receptor encoded by the second nucleotidesequence may specifically bind to the cancer cells of a cancer patient.The cancer patient may be subsequently treated with the modified Tcells. Suitable cancer patients for treatment with the modified T cellsmay be identified by a method comprising;

-   -   obtaining sample of cancer cells from an individual with cancer        and;    -   identifying the cancer cells as binding to the antigen receptor        encoded by the second nucleotide sequence and expressed by the        modified T cells.

Cancer cells may be identified as binding to the antigen receptorencoded by the second nucleotide sequence by identifying one or moretumour antigens expressed by the cancer cells. Methods of identifyingantigens on the surface of cancer cells obtained from an individual withcancer are well-known in the art.

In some embodiments, a heterologous antigen receptor suitable for thetreatment of a specific cancer patient may be identified by;

-   -   obtaining sample of cancer cells from an individual with cancer        and;    -   identifying an antigen receptor that specifically binds to the        cancer cells.

An antigen receptor that specifically binds to the cancer cells may beidentified for example by identifying one or more tumour antigensexpressed by the cancer cells. Methods of identifying antigens on thesurface of cancer cells obtained from an individual with cancer arewell-known in the art. An antigen receptor which binds to the one ormore tumour antigens or which binds to MHC-displayed peptide fragmentsof the one or more antigens may then be identified, for example fromantigen receptors of known specificities or by screening a panel orlibrary of antigen receptors with diverse specificities. Antigenreceptors that specifically bind to cancer cells having one or moredefined tumour antigens may be produced using routine techniques.

Nucleic acid encoding the identified antigen receptor may be used as thesecond nucleotide sequence in a nucleic acid construct as describedherein.

The cancer cells of an individual suitable for treatment as describedherein may express the antigen and may be of correct HLA type to bindthe antigen receptor.

Cancer cells may be distinguished from normal somatic cells in anindividual by the expression of one or more antigens (i.e. tumourantigens). Normal somatic cells in an individual may not express the oneor more antigens or may express them in a different manner, for exampleat lower levels, in different tissue and/or at a different developmentalstage. Tumour antigens may elicit immune responses in the individual. Inparticular, a tumour antigen may elicit a T cell-mediated immuneresponse against cancer cells in the individual that express the tumourantigen. One or more tumour antigens expressed by cancer cells in apatient may be selected as a target antigen for heterologous receptorson modified T cells.

Tumour antigens expressed by cancer cells may include, for example,cancer-testis (CT) antigens encoded by cancer-germ line genes, such asMAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,MAGE-A9, MAGE-A10, MACE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33/MUM-1, PRAME, NAG,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1/CT7,MAGE-C2, NY-ESO-I, LACE-I, SSX-I, SSX-2 (HOM-MEL-40), SSX-3, SSX-4,SSX-5, SCP-I and XAGE and immunogenic fragments thereof (Simpson et al.Nature Rev (2005) 5, 615-625, Gure et al., Clin Cancer Res (2005) 11,8055-8062; Velazquez et al., Cancer Immun (2007) 7, 11; Andrade et al.,Cancer Immun (2008) 8, 2; Tinguely et al., Cancer Science (2008);Napoletano et al., Am J of Obstet Gyn (2008) 198, 99 e91-97).

Other tumour antigens include, for example, overexpressed, upregulatedor mutated proteins and differentiation antigens particularly melanocytedifferentiation antigens such as p53, ras, CEA, MUC1, PMSA, PSA,tyrosinase, Melan-A, MART-1, gp100, gp75, alpha-actinin-4, Bcr-Ablfusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1,dek-can fusion protein, EF2, ETV6-AML1 fusion protein,LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2,KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9,pml-RAR.alpha. fusion protein, PTPRK, K-ras, N-ras, Triosephosphateisomerase, GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I),E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, alpha.-fetoprotein, 13HCG, BCA225, BTAA,CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1,CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag,MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 bindingprotein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS andtyrosinase related proteins such as TRP-1, TRP-2.

Other tumour antigens include out-of-frame peptide-MHC complexesgenerated by the non-AUG translation initiation mechanisms employed by“stressed” cancer cells (Malarkannan et al. Immunity 1999 June;10(6):681-90).

Other tumour antigens are well-known in the art (see for exampleWO00/20581; Cancer Vaccines and Immunotherapy (2000) Eds Stern, Beverleyand Carroll, Cambridge University Press, Cambridge) The sequences ofthese tumour antigens are readily available from public databases butare also found in WO 1992/020356 A1, WO 1994/005304 A1, WO 1994/023031A1, WO 1995/020974 A1, WO 1995/023874 A1 and WO 1996/026214 A1.

Preferred tumour antigens include NY-ESO1, PRAME, alpha-fetoprotein(AFP), MAGE A4, MAGE A1, MAGE A10 and MAGE B2, most preferably NY-ESO-1and MAGE-A10.

NY-ESO-1 is a human tumour antigen of the cancer/testis (CT) family andis frequently expressed in a wide variety of cancers, includingmelanoma, prostate, transitional cell bladder, breast, lung, thyroid,gastric, head and neck, and cervical carcinoma (van Rhee F. et al. Blood2005; 105(10): 3939-3944). In addition, expression of NY-ESO-1 isusually limited to germ cells and is not expressed in somatic cells(Scanlan M. J. et al. Cancer Immun. 2004; 4(1)). Suitable affinityenhanced TCRs that bind to cancer cells expressing NY-ESO-1 includeNY-ESO-1^(c259).

NY-ESO-1 c²⁵⁹ is an affinity enhanced TCR is mutated at positions 95 and96 of the alpha chain 95:96LY relative to the wildtype TCR. NY-ESO-1c²⁵⁹ binds to a peptide corresponding to amino acid residues 157-165 ofthe human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of theHLA-A2+ class 1 allele with increased affinity relative to theunmodified wild type TCR (Robbins et al J Immunol (2008) 180(9):6116).

MAGE-A10 is a highly immunogenic member of the MAGE-A family of CTantigens, and is expressed in germ cells but not in healthy tissue.MAGE-A10 is expressed in high percentages of cancer cells from a numberof tumours (Schultz-Thater E. et al. Int J Cancer. 2011;129(5):1137-1148).

The introduction of the nucleic acid construct into T cells and theirsubsequent expansion may be performed in vitro and/or ex vivo.

The first nucleotide sequence encoding the bioactive molecule and thesecond nucleotide sequence encoding the antigen receptor are introducedinto the T cells in the same expression vector. This increases theproportion of T cells which express both genes after transduction.

The first nucleotide sequence may be configured for expression in afirst direction and the second nucleotide sequence may be configured forexpression in a second direction in the nucleic acid construct. Forexample, the first nucleotide sequence encoding IL-7 may be in theforward orientation in the nucleic acid construct and the secondnucleotide sequence encoding the antigen receptor may be in the reverseorientation or the first nucleotide sequence encoding IL-7 may be in thereverse orientation and the second nucleotide sequence encoding theantigen receptor may be in the forward orientation. The first nucleotidesequence may be transcribed from the sense strand of the nucleic acidconstruct and the second nucleotide sequence may be transcribed from theantisense strand of the nucleic acid construct or the first nucleotidesequence may be transcribed from the antisense strand of the nucleicacid construct and the second nucleotide sequence may be transcribedfrom the sense strand of the nucleic acid construct.

The nucleic acid construct may include one or more unique restrictionsites to facilitate further manipulation.

In some embodiments, the nucleic acid construct may be introduceddirectly until T cells using gene editing techniques.

In other embodiments, the nucleic acid construct may be incorporatedinto an expression vector. Suitable vectors are well known in the artand are described in more detail herein.

Examples of suitable vectors include AB1581 and ADB967 as describedbelow. Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator fragments, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Preferably, the vectorcontains appropriate regulatory sequences to drive the expression of thenucleic acid in mammalian cells. A vector may also comprise sequences,such as origins of replication, promoter regions and selectable markers,which allow for its selection, expression and replication in bacterialhosts such as E. coli.

Preferably, the nucleic acid construct is contained in a viral vector,most preferably a gamma retroviral vector or a lentiviral vector, suchas a VSVg-pseudotyped lentiviral vector. The T cells may be transducedby contact with a viral particle comprising the nucleic acid. Viralparticles for transduction may be produced according to known methods.For example, HEK293T cells may be transfected with plasmids encodingviral packaging and envelope elements as well as a lentiviral vectorcomprising the coding nucleic acid. A VSVg-pseudotyped viral vector maybe produced in combination with the viral envelope glycoprotein G of theVesicular stomatitis virus (VSVg) to produce a pseudotyped virusparticle

A viral vector, such as a lentivirus, may be contained in a viralparticle comprising the nucleic acid vector encapsulated by one or moreviral proteins. A viral particle may be produced by a method comprisingtransducing mammalian cells with a viral vector as described herein andone or more viral packaging and envelope vectors and culturing thetransduced cells in a culture medium, such that the cells producelentiviral particles that are released into the medium.

Following release of viral particles, the culture medium comprising theviral particles may be collected and, optionally the viral particles maybe concentrated.

Following production and optional concentration, the viral particles maybe stored, for example by freezing at −80° C. ready for use intransducing T cells.

The nucleic acid construct or vector may be introduced into the T cellsby any convenient method. When introducing or incorporating aheterologous nucleic acid into a T cell, certain considerations must betaken into account, well-known to those skilled in the art. The nucleicacid to be inserted should be assembled within a construct or vectorwhich contains effective regulatory elements which will drivetranscription in the T cell. Suitable techniques for transporting theconstructor vector into the T cell are well known in the art and includecalcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or lentivirus. For example, solid-phasetransduction may be performed without selection by culture onretronectin-coated, retroviral vector-preloaded tissue culture plates.

Many known techniques and protocols for manipulation and transformationof nucleic acid, for example in preparation of nucleic acid constructs,introduction of DNA into cells and gene expression are described indetail in Protocols in Molecular Biology, Second Edition, Ausubel et al.eds. John Wiley & Sons, 1992.

Following the introduction of nucleic acid into the T cells, the initialpopulation of modified T cells may be cultured in vitro such that themodified T cells proliferate and expand the population.

The modified T cell population may for example be expanded usingmagnetic beads coated with anti-CD3 and anti-CD28. The modified T cellsmay be cultured using any convenient technique to produce the expandedpopulation. Suitable culture systems include stirred tank fermenters,airlift fermenters, roller bottles, culture bags or dishes, and otherbioreactors, in particular hollow fibre bioreactors. The use of suchsystems is well-known in the art.

Numerous culture media suitable for use in the proliferation of T cellsex vivo are available, in particular complete media, such as AIM-V,Iscoves medium and RPMI-1640 (Invitrogen-GIBCO). The medium may besupplemented with other factors such as serum, serum proteins andselective agents. For example, in some embodiments, RPMI-1640 mediumcontaining 2 mM glutamine, 10% FBS, 25 mM HEPES, pH 7.2, 1%penicillin-streptomycin, and 55 μM β-mercaptoethanol and optionallysupplemented with 20 ng/ml recombinant IL-2 may be employed. The culturemedium may be supplemented with the agonistic or antagonist factorsdescribed above at standard concentrations which may readily bedetermined by the skilled person by routine experimentation.

Conveniently, cells are cultured at 37° C. in a humidified atmospherecontaining 5% CO₂ in a suitable culture medium.

Methods and techniques for the culture of T cells and other mammaliancells are well-known in the art (see, for example, Basic Cell CultureProtocols, C. Helgason, Humana Press Inc. U.S. (15 Oct. 2004) ISBN:1588295451; Human Cell Culture Protocols (Methods in Molecular MedicineS.) Humana Press Inc., U.S. (9 Dec. 2004) ISBN: 1588292223; Culture ofAnimal Cells: A Manual of Basic Technique, R. Freshney, John Wiley &Sons Inc (2 Aug. 2005) ISBN: 0471453293, Ho W Y et al J Immunol Methods.(2006) 310:40-52)

In some embodiments, it may be convenient to isolate and/or purify themodified T cells from the population. Any convenient technique may beused, including FACS and antibody coated magnetic particles.

Optionally, the population of modified T cells produced as describedherein may be stored, for example by lyophilisation and/orcryopreservation, before use.

A population of modified T cells may be admixed with other reagents,such as buffers, carriers, diluents, preservatives and/orpharmaceutically acceptable excipients. Suitable reagents are describedin more detail below. A method described herein may comprise admixingthe population of modified T cells with a pharmaceutically acceptableexcipient.

Pharmaceutical compositions suitable for administration (e.g. byinfusion), include aqueous and non-aqueous isotonic, pyrogen-free,sterile injection solutions which may contain anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. Examples of suitable isotonic vehicles foruse in such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Suitable vehicles can be foundin standard pharmaceutical texts, for example, Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton,Pa., 1990.

In some preferred embodiments, the modified T cells may be formulatedinto a pharmaceutical composition suitable for intravenous infusion intoan individual.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgement, suitable for use in contactwith the tissues of a subject (e.g., human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Other aspects of the invention provide a population of modified T cellsexpressing a nucleic construct or a vector as described herein and apopulation of T cells that constitutively express a heterologous antigenreceptor and express IL-7 inducibly upon T cell activation.

The T cells may bind specifically to cancer cells. A suitable populationmay be produced by a method described above.

The population of modified T cells may be for use as a medicament. Forexample, a population of modified T cells as described herein may beused in cancer immunotherapy therapy, for example adoptive T celltherapy.

Other aspects of the invention provide the use of a population ofmodified T cells as described herein for the manufacture of a medicamentfor the treatment of cancer, a population of modified T cells asdescribed herein for the treatment of cancer, and a method of treatmentof cancer may comprise administering a population of modified T cells asdescribed herein to an individual in need thereof.

The population of modified T cells may be autologous i.e. the modified Tcells were originally obtained from the same individual to whom they aresubsequently administered (i.e. the donor and recipient individual arethe same). A suitable population of modified T cells for administrationto the individual may be produced by a method comprising providing aninitial population of T cells obtained from the individual, modifyingthe T cells to inducibly express IL-7 and constitutively express anantigen receptor which binds specifically to cancer cells in theindividual as described herein, and culturing the modified T cells.

The population of modified T cells may be allogeneic i.e. the modified Tcells were originally obtained from a different individual to theindividual to whom they are subsequently administered (i.e. the donorand recipient individual are different). The donor and recipientindividuals may be HLA matched to avoid GVHD and other undesirableimmune effects. A suitable population of modified T cells foradministration to a recipient individual may be produced by a methodcomprising providing an initial population of T cells obtained from adonor individual, modifying the T cells to inducibly express IL-7 andconstitutively express an antigen receptor which binds specifically tocancer cells in the recipient individual, as described herein, andculturing the modified T cells.

Following administration of the modified T cells, the recipientindividual may exhibit a T cell mediated immune response against cancercells in the recipient individual. This may have a beneficial effect onthe cancer condition in the individual.

Cancer conditions may be characterised by the abnormal proliferation ofmalignant cancer cells and may include leukaemias, such as AML, CML, ALLand CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma andmultiple myeloma, and solid cancers such as sarcomas, skin cancer,melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer,ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervicalcancer, liver cancer, head and neck cancer, oesophageal cancer, pancreascancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer,cancer of the gall bladder and biliary tracts, thyroid cancer, thymuscancer, cancer of bone, and cerebral cancer, as well as cancer ofunknown primary (CUP).

Cancer cells within an individual may be immunologically distinct fromnormal somatic cells in the individual (i.e. the cancerous tumour may beimmunogenic). For example, the cancer cells may be capable of elicitinga systemic immune response in the individual against one or moreantigens expressed by the cancer cells. The tumour antigens that elicitthe immune response may be specific to cancer cells or may be shared byone or more normal cells in the individual.

An individual suitable for treatment as described above may be a mammal,such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan,gibbon), or a human.

In preferred embodiments, the individual is a human. In other preferredembodiments, non-human mammals, especially mammals that areconventionally used as models for demonstrating therapeutic efficacy inhumans (e.g. murine, primate, porcine, canine, or rabbit animals) may beemployed.

In some embodiments, the individual may have minimal residual disease(MRD) after an initial cancer treatment.

An individual with cancer may display at least one identifiable sign,symptom, or laboratory finding that is sufficient to make a diagnosis ofcancer in accordance with clinical standards known in the art. Examplesof such clinical standards can be found in textbooks of medicine such asHarrison's Principles of Internal Medicine, 15th Ed., Fauci A S et al.,eds., McGraw-Hill, New York, 2001. In some instances, a diagnosis of acancer in an individual may include identification of a particular celltype (e.g. a cancer cell) in a sample of a body fluid or tissue obtainedfrom the individual.

Treatment may be any treatment and therapy, whether of a human or ananimal (e.g. in veterinary applications), in which some desiredtherapeutic effect is achieved, for example, the inhibition or delay ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, amelioration of the condition,cure or remission (whether partial or total) of the condition,preventing, delaying, abating or arresting one or more symptoms and/orsigns of the condition or prolonging survival of a subject or patientbeyond that expected in the absence of treatment.

Treatment may also be prophylactic (i.e. prophylaxis). For example, anindividual susceptible to or at risk of the occurrence or re-occurrenceof cancer may be treated as described herein. Such treatment may preventor delay the occurrence or re-occurrence of cancer in the individual.

In particular, treatment may include inhibiting cancer growth, includingcomplete cancer remission, and/or inhibiting cancer metastasis. Cancergrowth generally refers to any one of a number of indices that indicatechange within the cancer to a more developed form. Thus, indices formeasuring an inhibition of cancer growth include a decrease in cancercell survival, a decrease in tumour volume or morphology (for example,as determined using computed tomographic (CT), sonography, or otherimaging method), a delayed tumour growth, a destruction of tumourvasculature, improved performance in delayed hypersensitivity skin test,an increase in the activity of T cells, and a decrease in levels oftumour-specific antigens. Administration of T cells modified asdescribed herein may improve the capacity of the individual to resistcancer growth, in particular growth of a cancer already present thesubject and/or decrease the propensity for cancer growth in theindividual.

The modified T cells or the pharmaceutical composition comprising themodified T cells may be administered to a subject by any convenientroute of administration, whether systemically/peripherally or at thesite of desired action, including but not limited to; parenteral, forexample, by infusion. Infusion involves the administration of the Tcells in a suitable composition through a needle or catheter. Typically,T cells are infused intravenously or subcutaneously, although the Tcells may be infused via other non-oral routes, such as intramuscularinjections and epidural routes. Suitable infusion techniques are knownin the art and commonly used in therapy (see, e.g., Rosenberg et al.,New Eng. J. of Med., 319:1676, 1988).

Typically, the number of cells administered is from about 10⁵ to about10¹⁰ per Kg body weight, typically 2×10⁸ to 2×10¹⁰ cells per individual,typically over the course of 30 minutes, with treatment repeated asnecessary, for example at intervals of days to weeks. It will beappreciated that appropriate dosages of the modified T cells, andcompositions comprising the modified T cells, can vary from patient topatient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects of the treatments of the present invention. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular cells, the route ofadministration, the time of administration, the rate of loss orinactivation of the cells, the duration of the treatment, other drugs,compounds, and/or materials used in combination, and the age, sex,weight, condition, general health, and prior medical history of thepatient. The amount of cells and the route of administration willultimately be at the discretion of the physician, although generally thedosage will be to achieve local concentrations at the site of actionwhich achieve the desired effect without causing substantial harmful ordeleterious side-effects.

While the modified T cells may be administered alone, in somecircumstances the modified T cells may be administered cells incombination with the target antigen, APCs displaying the target antigen,and/or IL-2 to promote expansion in vivo of the population of modified Tcells. The population of modified T cells may be administered incombination with one or more other therapies, such as cytokines e.g.IL-2, cytotoxic chemotherapy, radiation and immuno-oncology agents,including checkpoint inhibitors, such as anti-B7-H3, anti-B7-H4,anti-TIM3, anti-KIR, anti-LAG3, anti-PD-1, anti-PD-L1, and anti-CTLA4antibodies.

The one or more other therapies may be administered by any convenientmeans, preferably at a site which is separate from the site ofadministration of the modified T cells.

Administration of modified T cells can be effected in one dose,continuously or intermittently (e.g., in divided doses at appropriateintervals) throughout the course of treatment. Methods of determiningthe most effective means and dosage of administration are well known tothose of skill in the art and will vary with the formulation used fortherapy, the purpose of the therapy, the target cell being treated, andthe subject being treated. Single or multiple administrations can becarried out with the dose level and pattern being selected by thetreating physician. Preferably, the modified T cells are administered ina single transfusion of a least 1×10⁹ T-cells.

The features disclosed in the foregoing description, or in the followingclaims, or in the accompanying drawings, expressed in their specificforms or in terms of a means for performing the disclosed function, or amethod or process for obtaining the disclosed results, as appropriate,may, separately, or in any combination of such features, be utilised forrealising the invention in diverse forms thereof.

It is to be understood that the application discloses all combinationsof any of the above aspects and embodiments described above with eachother, unless the context demands otherwise. Similarly, the applicationdiscloses all combinations of the preferred and/or optional featureseither singly or together with any of the other aspects, unless thecontext demands otherwise.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

Modifications of the above embodiments, further embodiments andmodifications thereof will be apparent to the skilled person on readingthis disclosure, and as such, these are within the scope of the presentinvention.

All documents and sequence database entries mentioned in thisspecification are incorporated herein by reference in their entirety forall purposes.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Other aspects and embodiments of the invention provide the aspects andembodiments described above with the term “comprising” replaced by theterm “consisting of” and the aspects and embodiments described abovewith the term “comprising” replaced by the term “consisting essentiallyof”.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described above.

Experiments

1. Methods

1.1 Design and Production of IL-7 Expression Constructs

A lentiviral expression vector was designed that permitted the inducibleexpression of IL-7 following recognition of the cognate peptide MHC1complex. The inducible element was based on NFAT/AP1 transcriptionalresponse element (TRE) that is present in the human IL2 promoter (Macianet al., 2001 Oncogene). The selective inducible expression of IL-7 is aresult of TCR activation, and Ca²⁺ dependent translocation of NFAT tothe nucleus where it can promote transcription of genes which harbour anNFAT TRE.

The IL-7 expression cassette was is composed of four elements asillustrated in FIG. 1.

-   -   1. 3 tandem repeats of the human IL2 promoter NFAT TRE        highlighted in red

(GGAGGAAAAACTGTTTCATACAGAAGGCGT)

-   -   2. Minimal CMV promoter (green)    -   3. Kozak sequence (GCCGCCACCATG) and codon optimised IL-7 coding        sequence (Yellow). Supplementary FIG. 1 contains the wild-type        nucleotide sequence    -   4. SV40 polyadenylation signal (purple)

The sequence upstream of the EcorV restriction (highlighted in green inFIG. 1) corresponds to the lentiviral vector backbone sequence and wasincluded in the synthetic construct in order to maintain the codingsequence of the EF1A promoter. Additional unique restriction sites werealso designed into the synthetic construct in order to permit furtherengineering of the construct should it be required. These include:

-   -   1. EcoRV (GATATC) and NSI-I (ATGCAT) restriction sites upstream        of the NFAT IL-2 TRE    -   2. NdeI restriction site CATATG immediately upstream of the IL-7        CDS    -   3. XhoI (CTCGAG) immediately following the TGA stop codon in the        IL-7 CDS    -   4. AatII restriction site (GACGTC) immediately upstream of the        mCMV promoter.

This construct was designed to be cloned between the AgeI and MluIrestriction sites within a construct expressing a TCR alone (FIG. 2) andwill permit transcription of the IL-7 from the antisense strand and inreverse orientation to the SPEAR TCR. Additional constructs weredesigned that permitted the constitutive expression of IL-7 and theSPEAR TCR. These constructs encode IL-7 and the TCRα and TCRβ chains asa single open reading frame. The individual polypeptides are separatedby self-cleaving 2A-like peptides that permit ribosomal skipping and theexpression of multiple polypeptides from a single transcript.

1.2 Generation of ADB967 NFAT IL-7_TCR1

The NFAT_IL-7 cassette (SEQ ID NO: 1) was synthesised synthetically byGeneArt (Thermo Fisher Scientific) with 5′ AgeI (ACCGGT) 3′ MluI(ACGCGT) restriction sites and was provided in a standard cloning vector(pMS-RQ) (FIG. 2). The NFAT_IL-7 containing vectors and ADB934 (TCR1)were digested with AgeI and MluI and the fragments of interest werepurified by gel extraction using a NucleoSpin® gel extraction kitaccording to manufacturer's instruction. Digested fragments were ligatedusing T4 DNA ligase (NEB) according to standard protocols. Clones werescreened by restriction enzyme digest and verified by sequencing (seethe vector map of FIG. 4).

1.3 Generation of ADB1099 Constitutive IL-7_TCR1

The IL-7_T2A_TCR1 insert was generated by overlapping PCR. The IL-7 CDSwas amplified with primers NHEI_IL_7 F and IL7_T2AR. The TCR1 CDS wasamplified with the primers T2AF and BETA_SAL_REVIII. The TCR1 CDS wasamplified from an existing in-house construct ADB951. PCR reactions wereperformed with Q5 DNA polymerase (NEB) according to standard protocolsand PCR products were purified by gel extraction using a NucleoSpin® gelextraction kit according to manufacturer's instruction. The twofragments were fused together by overlapping PCR with the NHEI_IL_7 Fand BETA_SAL_REVIII primers which contain NheI (GCTAGC) and SalI(GTCGAC) restriction sites (underlined). Digested fragments were ligatedusing T4 DNA ligase (NEB) according to standard protocols. Clones werescreened by restriction enzyme digest and verified by sequencing (seethe vector map of FIG. 5).

Primer Sequence NHEI_IL_7F TAATGCTAGCGCCGCCACCATGTTCCACG IL7T2ARCAGCAGGCTGCCTCTGCCCTCGCCAGAGCCGCTTCTCTTGGCTCTGCTTCCGTGCTCTTTGGTGCCCATCAGG T2AF AGGGCAGAGGCAGCCTGCTG BETA_SAL_ATTATTGTCGACTTAGCCCCGGCTGTCCTTCCGCTTCACC REVIII

1.4 Generation of ADB1581 NFATIL-7_TCR2

The TCR2 insert was subcloned from in-house construct ADB1535 intoADB1224. ABD1535 and ADB1224 were digested with NheI and SalIrestriction enzymes and fragments of interest were purified by gelextraction using a NucleoSpin® gel extraction kit according tomanufacturer's instruction. Digested fragments were ligated using T4 DNAligase (NEB) according to standard protocols. The NFAT IL-7 cassette wassubcloned from ADB967 (NFAT-IL-7_TCR1) into this new TCR2 lentiviralexpression vector between the AgeI and MluI restriction sites. Cloneswere screened by restriction enzyme digest and verified by sequencing(see the vector map of FIG. 6).

1.5 Generation of ADB1580 Constitutive IL-7_F2A_TCR2

The IL-7 CDS was PCR amplified from ABD1099 with the following primers

FWD- 5′ TAATGCTAGCGCCGCCACCATGTTCCACGTGTCC 3′ and Rev5′-CTTCACGGGCGCGCCAGAGCCGCTTCTCTTGG-3′.These include NheI (GCTAGC) and AscI (GGCGCGCC) restriction sites(underlined). PCR products were purified by gel extraction and purifiedby gel extraction using a NucleoSpin® gel extraction kit according tomanufacturer's instruction. The PCR product and (ADB1488) were digestedwith NheI and AscI and fragments of interest purified by gel extractionusing a NucleoSpin® gel extraction kit according to manufacturer'sprotocol. Digested fragments were ligated using T4 DNA ligase (NEB)according to standard protocols. Clones were screened by restrictionenzyme digest and verified by sequencing (see the vector map of FIG. 7).

1.6 T cell Production

PBLs from six donors were separated from whole blood with additionalCD14 depletion (PBL) using MACS isolation kits and associatedmanufacturer's protocols. T cells were transduced with differentlentiviral constructs including: TCR and NFAT_IL-7 TCR (see FIG. 1). Tcell transductions were performed in the presence of 1 mg/ml F108Poloxamer. The F108 Poloxamer had previously been dissolved in water tomake a stock solution at 100 mg/ml and was then sterilised using a 0.2μm filter before storing at 4° C. 1 mg/ml F108 Poloxamer was added toeach well of cells (including NTD cells) at the same time as addition ofthe lentiviral particles.

Transduced cells were expanded for 14 days. T cells were fed with100U/ml Proleukin on day 10 but fed with fresh media without Proleukinon day 12 to allow the cells more time to rest prior to use for assays,particularly for proliferation assays.

On day 14 cells were frozen. Non-transduced (NTD) T cells were producedat the same time as the transduced T cells but fresh virus medium(R10+Hepes) was added to the stimulated T cells in place of lentiviralparticles.

1.7 Cytokine Secretion Assays for ELISA

Cytokine production assays were typically carried out as follows. On theday of the assay, A375, Mel624 and Colo205 cells were harvested,resuspended in R10 and counted. Some of the A375 cells were pulsed for 2hours at 37° C./5% CO₂ with 10 μM MAGE-A4 peptide, before washing threetimes. Target cells were typically plated out at 50,000 cells/well in avolume of 100 μl into 96 well flat bottom plates in triplicate wells. Tcells were thawed, washed, resuspended in R10 at no less than 1×10⁶cells/ml and rested for 1-2h at 37° C./5% CO₂. Non-transduced andtransduced effector cells were counted and were plated at 120,000 cellsper well in 100 μl assay medium, in triplicate wells, to give a finalvolume of 200 μl. Note: to account for differences in transductionbetween the different constructs, the cells were normalised by addingNTD T cells from the same donor to give equivalent transductionefficiencies. Plates were then put in the incubator at 37° C./5% CO₂ for48 hours. After 48 hours, the assay plates were centrifuged andsupernatant transferred to new 96 well plates, which were stored at −20°C. until analysis by ELISA which was performed at a later date.

1.8 IL-7 ELISA

This assay was performed in 96 well half area plates using the DuosetHuman IL-7 ELISA kit according to the manufacturer's protocol withmodifications. Briefly, as the 96 well half area plates were used, 25μl/well (or 50 μl of blocking reagent) was used for all reagents insteadof 100 μl as indicated in the protocol. In most cases the samples wereused undiluted. If they were diluted they were diluted in assay medium.The assays were developed using commercial TMB substrate solution forapprox. 10 minutes and the reaction stopped with 1N H2SO4. Assays wereread using the Spectrastar Omega at 450 nm with wavelength correctionset to 540 nm. Data were analysed using Spectrastar data analysissoftware. Data points greater than the top concentration on the standardcurve were normally allocated the value of the top concentration. Formost assays the standard curve was extended to provide a greater rangethan indicated by the manufacturer, with a top concentration of 1000pg/ml. Samples and standard curves were either performed using duplicateor triplicate wells and the curve fitting was performed using fourparameter logistic curve fitting where possible.

1.9 T Cell Phenotyping

On the day of the assay, the T cells were thawed, washed twice in PBS,counted and adjusted to 1×10⁶ cells/ml in PBS. Flow cytometry wasperformed on T cells. The list of antibodies used can be found in Tables1 and 2.

TABLE 1 List of antibodies and volumes used to identify proliferating Tcells at the end of the expansion phase (Panel 1) Antibody Volume/test(μl) CD45RA-PE Cy7 (BioLgend: 304126) 1.25 μl CD45RO-PerCP Cy5.5(BioLegend: 304222) 1.25 μl CCR7-PE CF594 (BD Bioscience: 562381) 5 μlVα24-PE (Beckman Coulter: IM2283) 20 PD-1-BV786 (BioLegend: 329929) 2.5μl CD4-BV650 (BD Bioscience: 56875) 2.5 μl CD8-APC-ef780 (eBioscience:47-0087-42) 2.5 μl CD3-FITC (eBioscience: 11-0036-42) 2.5 μl TIM-3-APC(R&D Systems: FAB2365A) 5 ul IL7Rα (CD127)-BV421 (BioLegend: 47-1278-42)5 ul LIVE/DEAD ® Fixable Aqua Dead Cell 1/800 dilution Stain (LifeTechnologies: L34957)

TABLE 2 List of antibodies and volumes used to identify proliferating Tcells at the end of the expansion phase (Panel 2) Antibody Volume/test(μl) CD8-APC-ef780 (eBioscience: 47-0087-42) 2.5 μl CD4-BV650 (BDBioscience: 56875) 2.5 μl CD45RA-PE Cy7 (BioLgend: 304126) 1.25 μlCCR7-PE CF594 (BD Bioscience: 562381) 5 μl Vα24-PE (Beckman Coulter:IM2283) 20 OX-40 (CD134)-FITC (BD Bioscience: 555837) 5 μl CD40L(CD154)-BV421 (BD Bioscience: 563886) 5 μl CD28-PerCPCy5.5 (BDBioscience: 560685) 2.5 μl CD27-BV786 (BD Bioscience: 563327) 5 μl4-1BB-APC (BD Bioscience: 550890) 2.5 μl LIVE/DEAD ® Fixable Aqua DeadCell 1/800 dilution Stain (Life Technologies: L34957)

1.10 Restimulation Assay

The restimulation assays were carried out as follows. On the first dayof the assay (day 0) T cells were thawed, washed, resuspended in R10 atno less than 1×10⁶ cells/ml and rested.

Cells were counted and in order to account for differences intransduction between the different lentiviral preparations, the cellswere normalised by adding NTD T cells from the same donor to giveequivalent transduction efficiencies within a batch of donor T cells.

T cells were plated out in R10 at 1×10⁶/ml, with 1 ml of T cells perwell in a 24 well plate. A375 target cells were harvested and thenirradiated (48 Gy). A375 cells were resuspended at approximately1×10⁶/ml and 1 ml per well added to the wells containing T cells. Thisgave a final volume of 2 ml/well with 1:1 T cells and irradiated A375cells. In control wells recombinant human IL-7 was added to the cells ata final concentration of 20 ng/ml. Co-cultures of T cells and irradiatedA375 cells with or without exogenous IL-7 were then cultured for 7 daysat 37° C./5% CO₂.

On days 7, 14, 21 and 28, T cells were harvested and counted usingsemi-automated counting with Trypan Blue positive cells (dead cells)excluded. For wells containing T cells that did not express the TCR1,not all the Trypan Blue-negative (live) cells were T cells, as they weremuch larger than T cells. These were most likely to be irradiated A375targets that were not yet positive for Trypan Blue staining. As many aspossible of the larger, obviously non-T cells (A375) were manuallyexcluded from the counts but live T cell counts from those wells(containing NTD) are likely to be an overestimate as they stillcontained some “apparently” live A375 targets. This was also reflectedby the larger average size of the cells from those samples as A375 cellsare larger than T cells. However in wells containing T cells expressingthe TCR1 there were few or no large, live A375 cells remaining, soabsolute counts and cell sizes are likely to be more accurate. The totallive T cell count was then calculated.

Once the total number of live T cells had been calculated, T cells weresplit as follows. If the T cells had proliferated, they were diluted toa concentration of 1×10⁶/ml (to the nearest ml) by adding fresh R10 tothe existing volume of T cells (in “old” R10) to the correct finalconcentration. “Old” R10 is defined as the media that the cells had beencultured in for the previous week. If however the T cells had contracted(i.e. the number of live T cells had reduced compared with theconcentration originally plated out), cells were centrifuged (1500 rpm,5 minutes) and then resuspended in the appropriate volume of “old” R10.

T cells were plated out at 1 ml/well (˜1×10⁶ T cells/ml) in 24 wellplates. Where possible, all the T cells were plated out as theyexpanded, so that the total number of T cells presented in the figureswas actually the total number of T cells produced. In some instances,particularly at later time points as the number of T cells grewincreasingly large (usually greater than 8-12×10⁶ cells), only 8-12×10⁶T cells were plated out for restimulation. The total number of T cellsfor those samples at subsequent time-points was then re-calculated byfactoring in the number of T cells that had been originally plated outat the previous time-point. For example, if we had counted 20×10⁶ Tcells on day 21, we would only plate out 8×10⁶ of those cells on day 21for restimulation. The total cell numbers on day 28, which would havebeen generated if we had plated out all 20×106 cells, would bere-calculated as follows:

-   1. Fold change in cell numbers=Cell count after restimulation (on    day 28)/Cell number plated out for restimulation (8×10⁶ originally    plated out on day 21)-   2. Re-calculated total cell numbers on day 28=Fold change in cell    numbers (between days 21-28) x total number of cells that were    available for plating out on day 21 (e.g. 20×10⁶)

A375 target cells were harvested and irradiated at 48 Gy to preventtheir proliferation, and then added to the T cells at approximately1×10⁶/ml, 1 ml/well (1:1 T cell: A375) in R10. In control wellsrecombinant human IL-7 was added to the cells at a final concentrationof 20 μg/ml (assuming that all the IL-7 had been “consumed” in thewell). This restimulation process was repeated on days 7, 14 and 21.

1.11 In Vivo Studies

PBL from a Leukopak were depleted of CD14 using the methods previouslydescribed. T cells were transduced as previously described and expandedfor 14 days before freezing. In addition to the NTD T cells and T cellstransduced with constructs expressing TCR1, NFAT-IL-7_TCR1 orconstitutive IL-7_TCR1, T cells were also transduced with a constructexpressing NFAT-IL-7_irrelevant TCR.

On day 0, immunodeficient CIEA NOG(NOD.Cg-Prkdc_(scid)II2g_(tm1Sug)/JicTac) female mice aged 6-8 weekswere injected intravenously (i.v.) with 1×10⁶ Mel624 tumour cells thathad previously been transduced with a lentiviral construct expressingGFP/Luciferase. On day 6 post-tumour cell implantation, mice were imagedand randomised into 8 groups of 8 mice groups, based on luciferasesignal strength so that each group had an equal mean total flux. On day7, T cells were thawed and the frequency of transduced T cells betweenthe different constructs was normalised using NTD T cells to ˜42% TCRVβ+/CD3+. Mice were then i.v. injected either with nothing (tumour onlycontrol) or with 2×10⁶ total T cells (NTD, TCR1, NFAT-IL-7_TCR1,Constitutive IL-7_TCR1, NFAT-IL-7_control TCR).

Animals were imaged using the Bruker In Vivo Xtreme imaging system onday 6 and then once weekly to measure disease burden and follow diseaseprogression. At later stages in the study animals were imaged twiceweekly. Prior to imaging, animals were injected i.p. with 150 mg/kg ofluciferin (5 ml/kg) and anaesthetised with isoflurane. After theexposure an X-ray picture was taken to aid orientation and organpositioning. At the time of analysis, images were converted tophotons/second/mm² (P/s/mm²) which allows comparison of images acquiredusing different exposure times, the scale was adjusted and thebioluminescence image was superimposed upon the X-ray image. A region ofinterest was set to measure the bioluminescence signal from the wholeanimal. The size of the ROI was kept identical for all images. Mice werealso weighed at least 3 times per week and were culled when eitherweight loss or poor condition indicated

Results

Mice injected with T cells co-expressing inducible IL-7 with anengineered TCR were found to show improved survival compared with cellsexpressing TCR alone or TCR together with constitutive IL-7 (FIG. 16).

Sequences Nucleotide sequence of the NFAT IL-7 inducible cassette.SEQ ID NO: 1ACCGGTTCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGCGCTCTGCCCACTGACGGGCACCGGAGCCTCACGATGCATGATATCGGCCTAACTGGCCGGTACCTGAGCTCGCTAGCGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTga

CGCCACCATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCATCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGG

Key: Full underline = Tandem repeats of the human IL2 promoter NFAT TRE(GGAGGAAAAACTGTTTCATACAGAAGGCGT). Dashed underline =Minimal CMV promoter. No underline =The Kozak sequence (GCCGCCACCATG) and codon optimised IL-7 coding sequence. Dotted underline = The SV40 polyadenylation signal.Translated nucleotide sequence (SEQ ID NO: 2) and protein sequence (SEQ ID NO: 3) of codon optimised IL-7atgttccatgtttcttttaggtatatctttggacttcctcccctgatccttgttctgttg M  F  H  V  S  F  R  Y  I  F  G  L  P  P  L  I  L  V  L  Lccagtagcatcatctgattgtgatattgaaggtaaagatggcaaacaatatgagagtgtt P  V  A  S  S  D  C  D  I  E  G  K  D  G  K  Q  Y  E  S  Vctaatggtcagcatcgatcaattattggacagcatgaaagaaattggtagcaattgcctg L  M  V  S  I  D  Q  L  L  D  S  M  K  E  I  G  S  N  C  Laataatgaatttaacttttttaaaagacatatctgtgatgctaataaggaaggtatgttt N  N  E  F  N  F  F  K  R  H  I  C  D  A  N  K  E  G  M  Fttattccgtgctgctcgcaagttgaggcaatttcttaaaatgaatagcactggtgatttt L  F  R  A  A  R  K  L  R  Q  F  L  K  M  N  S  T  G  D  Fgatctccacttattaaaagtttcagaaggcacaacaatactgttgaactgcactggccag D  L  H  L  L  K  V  3  E  G  T  T  I  L  L  N  C  T  G  Qgttaaaggaagaaaaccagctgccctgggtgaagcccaaccaacaaagagtttggaagaa V  K  G  R  K  P  A  A  L  G  E  A  Q  P  T  K  S  L  E  Eaataaatctttaaaggaacagaaaaaactgaatgacttgtgtttcctaaagagactatta N  K  S  L  K  E  Q  K  K  L  N  D  L  C  F  L  K  R  L  Lcaagagataaaaacttgttggaataaaattttgatgggcactaaagaacactga Q  E  I  K  T  C  W  N  K  I  L  M  G  T  K  E  H  -IL-7 protein sequence SEQ ID NO: 3MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH*Nucleotide sequence between the Nhel and Sall sites SEQ ID NO: 4ATGGAGACCCTGCTGGGCCTGCTGATCCTGTGGCTGCAGCTCCAGTGGGTGTCCAGCAAGCAGGAGGTGACCCAGATCCCTGCCGCCCTGAGCGTGCCCGAGGGCGAGAACCTGGTGCTGAACTGCAGCTTCACCGACTCCGCCATCTACAACCTGCAGTGGTTCCGGCAGGACCCCGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCAGCCAGCGGGAGCAGACCAGCGGACGGCTGAACGCCAGCCTGGACAAGAGCAGCGGCCGGAGCACCCTGTACATCGCCGCCAGCCAGCCCGGCGACAGCGCCACCTACCTGTGCGCTGTGCGGCCTCTGTACGGCGGCAGCTACATCCCCACCTTCGGCAGAGGCACCAGCCTGATCGTGCACCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGTCTGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAATGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGCGGCTCCCGGGCCAAGAGAAGCGGATCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGAGACGTGGAAGAAAACCCTGGCCCTAGGATGAGCATCGGCCTGCTGTGCTGCGCCGCCCTGAGCCTGCTGTGGGCAGGACCCGTGAACGCCGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTATCGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACTCTGTGGGAGCCGGAATCACCGACCAGGGCGAGGTGCCCAACGGCTACAATGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCTGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGCAACACCGGCGAGCTGTTCTTCGGCGAGGGCTCCAGGCTGACCGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACACTGGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTCTACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGTCCTCCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATTGTGAGCGCCGAGGCCTGGGGCAGGGCCGACTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCCGGGGCTAAGTCGAC Protein sequence of TCR1 SEQ ID NO: 5METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*V TCR1 CDS SEQ ID NO: 6METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKD SRGSense strand 5′ to 3′ sequence spanning the region between the Mluland Sall restriction sites. SEQ ID NO: 7ACGCGTTAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTCTCGAGTCAGTGCTCTTTGGTGCCCATCAGGATCTTGTTCCAGCAGGTCTTGATTTCCTGCAGCAGCCGCTTCAGGAAGCACAGGTCGTTCAGTTTCTTCTGCTCTTTCAGGGACTTGTTCTCTTCCAGGCTCTTGGTAGGCTGGGCTTCTCCCAGGGCGGCAGGCTTTCTGCCCTTCACTTGGCCGGTGCAATTCAGCAGGATGGTGGTGCCCTCGGACACTTTCAGCAGATGCAGGTCGAAGTCGCCGGTGCTGTTCATCTTCAGGAACTGCCGCAGCTTTCTGGCGGCTCTGAACAGGAACATGCCTTCTTTGTTGGCGTCGCAGATGTGCCGCTTGAAGAAGTTGAACTCGTTGTTCAGGCAGTTGCTGCCGATTTCCTTCATGCTGTCCAGCAGCTGGTCGATGGACACCATCAGCACGCTCTCGTACTGCTTGCCGTCCTTGCCCTCGATGTCGCAGTCGCTGCTGGCCACAGGCAGCAGCACCAGGATCAGGGGGGGCAGGCCGAAGATGTACCGGAAGGACACGTGGAACATGGTGGCGGCCATATGGATCCAACGAATGTCGAGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGGCCTCCCACCGTACACGCCTAGACGTCACGCCTTCTGTATGAAACAGTTTTTCCTCCACGCCTTCTGTATGAAACAGTTTTTCCTCCACGCCTTCTGTATGAAACAGTTTTTCCTCCGCTAGCGAGCTCAGGTACCGGCCAGTTAGGCCGATATCATGCATCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGCCGCCACCATGGAGACCCTGCTGGGCCTGCTGATCCTGTGGCTGCAGCTCCAGTGGGTGTCCAGCAAGCAGGAGGTGACCCAGATCCCTGCCGCCCTGAGCGTGCCCGAGGGCGAGAACCTGGTGCTGAACTGCAGCTTCACCGACTCCGCCATCTACAACCTGCAGTGGTTCCGGCAGGACCCCGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCAGCCAGCGGGAGCAGACCAGCGGACGGCTGAACGCCAGCCTGGACAAGAGCAGCGGCCGGAGCACCCTGTACATCGCCGCCAGCCAGCCCGGCGACAGCGCCACCTACCTGTGCGCTGTGCGGCCTCTGTACGGCGGCAGCTACATCCCCACCTTCGGCAGAGGCACCAGCCTGATCGTGCACCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGTCTGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAATGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGCGGCTCCCGGGCCAAGAGAAGCGGATCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGAGACGTGGAAGAAAACCCTGGCCCTAGGATGAGCATCGGCCTGCTGTGCTGCGCCGCCCTGAGCCTGCTGTGGGCAGGACCCGTGAACGCCGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTATCGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACTCTGTGGGAGCCGGAATCACCGACCAGGGCGAGGTGCCCAACGGCTACAATGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCTGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGCAACACCGGCGAGCTGTTCTTCGGCGAGGGCTCCAGGCTGACCGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACACTGGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTCTACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGTCCTCCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATTGTGAGCGCCGAGGCCTGGGGCAGGGCCGACTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCCGGGGCTAAGTCGACNucleotide sequence of Constitutive-IL-7_T2A_TCR1. This sequence coversthe region between the Nhel and Sall restriction sites. SEQ ID NO: 8GCTAGCGCCGCCACCATGTTCCACGTGTCCTTCCGGTACATCTTCGGCCTGCCCCCCCTGATCCTGGTGCTGCTGCCTGTGGCCAGCAGCGACTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAAATCGGCAGCAACTGCCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAAGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGCTGCGGCAGTTCCTGAAGATGAACAGCACCGGCGACTTCGACCTGCATCTGCTGAAAGTGTCCGAGGGCACCACCATCCTGCTGAATTGCACCGGCCAAGTGAAGGGCAGAAAGCCTGCCGCCCTGGGAGAAGCCCAGCCTACCAAGAGCCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAACTGAACGACCTGTGCTTCCTGAAGCGGCTGCTGCAGGAAATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAAGAGCACGGAAGCAGAGCCAAGAGAAGCGGCTCTGGCGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAAAACCCTGGCCCTATGGAGACCCTGCTGGGCCTGCTGATCCTGTGGCTGCAGCTCCAGTGGGTGTCCAGCAAGCAGGAGGTGACCCAGATCCCTGCCGCCCTGAGCGTGCCCGAGGGCGAGAACCTGGTGCTGAACTGCAGCTTCACCGACTCCGCCATCTACAACCTGCAGTGGTTCCGGCAGGACCCCGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCAGCCAGCGGGAGCAGACCAGCGGACGGCTGAACGCCAGCCTGGACAAGAGCAGCGGCCGGAGCACCCTGTACATCGCCGCCAGCCAGCCCGGCGACAGCGCCACCTACCTGTGCGCTGTGCGGCCTCTGTACGGCGGCAGCTACATCCCCACCTTCGGCAGAGGCACCAGCCTGATCGTGCACCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGTCTGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAATGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGCGGCTCCCGGGCCAAGAGAAGCGGATCCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGAGACGTGGAAGAAAACCCTGGCCCTAGGATGAGCATCGGCCTGCTGTGCTGCGCCGCCCTGAGCCTGCTGTGGGCAGGACCCGTGAACGCCGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTATCGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACTCTGTGGGAGCCGGAATCACCGACCAGGGCGAGGTGCCCAACGGCTACAATGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCTGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGCAACACCGGCGAGCTGTTCTTCGGCGAGGGCTCCAGGCTGACCGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACACTGGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTCTACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGTCCTCCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATTGTGAGCGCCGAGGCCTGGGGCAGGGCCGACTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCCGGGGCTAAGTCGACProtein sequence of constitutive IL-7_TCR1 IL7 is underlined, TCR isitalicised. SEQ ID NO: 9MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHGSRAKRSGSGEGRGSLLTCGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Sense strand 5′ to 3′sequence spanning the region between the Mlul andSall restriction sites (see FIG. 6). SEQ ID NO: 10ACGCGTTAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTCTCGAGTCAGTGCTCTTTGGTGCCCATCAGGATCTTGTTCCAGCAGGTCTTGATTTCCTGCAGCAGCCGCTTCAGGAAGCACAGGTCGTTCAGTTTCTTCTGCTCTTTCAGGGACTTGTTCTCTTCCAGGCTCTTGGTAGGCTGGGCTTCTCCCAGGGCGGCAGGCTTTCTGCCCTTCACTTGGCCGGTGCAATTCAGCAGGATGGTGGTGCCCTCGGACACTTTCAGCAGATGCAGGTCGAAGTCGCCGGTGCTGTTCATCTTCAGGAACTGCCGCAGCTTTCTGGCGGCTCTGAACAGGAACATGCCTTCTTTGTTGGCGTCGCAGATGTGCCGCTTGAAGAAGTTGAACTCGTTGTTCAGGCAGTTGCTGCCGATTTCCTTCATGCTGTCCAGCAGCTGGTCGATGGACACCATCAGCACGCTCTCGTACTGCTTGCCGTCCTTGCCCTCGATGTCGCAGTCGCTGCTGGCCACAGGCAGCAGCACCAGGATCAGGGGGGGCAGGCCGAAGATGTACCGGAAGGACACGTGGAACATGGTGGCGGCCATATGGATCCAACGAATGTCGAGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGGCCTCCCACCGTACACGCCTAGACGTCACGCCTTCTGTATGAAACAGTTTTTCCTCCACGCCTTCTGTATGAAACAGTTTTTCCTCCACGCCTTCTGTATGAAACAGTTTTTCCTCCGCTAGCGAGCTCAGGTACCGGCCAGTTAGGCCGATATCATGCATCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGTGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGCTAGCCGCCACCATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTCAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGCGGCAGCCGGGCCAAGAGATCTGGATCCGGCGCTACCAACTTTAGCCTGCTGAAGCAGGCCGGGGACGTGGAAGAAAACCCTGGCCCTAGGATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGAGGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGCTAATAAGTCGAC TCR2 sequence SEQ ID NO: 11MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG Nucleotide sequence covering the region between the Nhel and Sallrestriction sites. SEQ ID NO: 12GCTAGCGCCGCCACCATGTTCCACGTGTCCTTCCGGTACATCTTCGGCCTGCCCCCCCTGATCCTGGTGCTGCTGCCTGTGGCCAGCAGCGACTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAAATCGGCAGCAACTGCCTGAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAAGAAGGCATGTTCCTGTTCAGAGCCGCCAGAAAGCTGCGGCAGTTCCTGAAGATGAACAGCACCGGCGACTTCGACCTGCATCTGCTGAAAGTGTCCGAGGGCACCACCATCCTGCTGAATTGCACCGGCCAAGTGAAGGGCAGAAAGCCTGCCGCCCTGGGAGAAGCCCAGCCTACCAAGAGCCTGGAAGAGAACAAGTCCCTGAAAGAGCAGAAGAAACTGAACGACCTGTGCTTCCTGAAGCGGCTGCTGCAGGAAATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAAGAGCACGGAAGCAGAGCCAAGAGAAGCGGCTCTGGCGCGCCCGTGAAGCAGACCCTGAACTTCGACCTGCTGAAACTGGCCGGCGACGTGGAAAGCAACCCTGGCCCTATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACTCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGCTGGTACAAGCAGGATACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCTCCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACTCCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGATATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCTAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAATTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGCGGCTGTGGTCCTCTGGCTCTCGGGCCAAGAGAAGCGGCAGCGGCGCCACCAATTTCAGCCTGCTGAAGCAGGCAGGGGATGTGGAAGAGAATCCCGGCCCTAGAATGGCCTCCCTGCTGTTTTTCTGCGGCGCCTTCTACCTGCTGGGGACCGGCAGCATGGACGCTGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCAGCCAGACAAAGGGCCACGACCGGATGTACTGGTACAGACAGGATCCAGGACTGGGCCTGAGGCTGATCTACTACAGCTTCGATGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCCCAGGCCAAGTTCTCCCTGAGCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGAGGAACAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCAGTGTTCGAGCCTAGCGAGGCCGAGATCTCCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGATTCTACCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAATCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGGGCCAAGCCCGTGACTCAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGCGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGCTGATGAGGTCGACTranslated protein sequence of constitutive IL-7_TCR2 SEQ ID NO: 13MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHGSRAKRSGSGAPVKQTLNFDLLKLAGDVESNPGPMKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG** human IL2 promoter NFAT TRE SEQ ID NO: 14GGAGGAAAAACTGTTTCATACAGAAGGCGT minimal CMV promoter SEQ ID NO: 15TAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCTCGACATTCGTTGGA TC

1. A method of treating cancer using immunotherapy, comprisingadministering to a patient in need thereof a population of modified Tcells comprising a nucleic acid construct that comprises: (i) a firstnucleotide sequence encoding a bioactive molecule, (ii) a secondnucleotide sequence encoding an antigen receptor; (iii) an induciblepromoter operably linked to the first nucleotide sequence and (iv) aconstitutive promoter operably linked to the second nucleotide.
 2. Themethod according to claim 1 wherein expression from the induciblepromoter is induced by activation of the modified T cells.
 3. The methodaccording to claim 1 wherein the inducible promoter comprises a nuclearfactor of activated T cells (NFAT) transcriptional response element(TRE).
 4. The method according to claim 3 wherein the NFAT TRE has thenucleic acid sequence of SEQ ID NO: 14 or a variant thereof.
 5. Themethod according to claim 3 wherein the inducible promoter comprisesthree or more copies of the NFAT TRE.
 6. The method according to claim 1wherein the nucleic acid construct comprises the sequence of SEQ IDNO:
 1. 7. The method according to claim 1 wherein the constitutivepromoter is the Human elongation factor-1 alpha promoter.
 8. The methodaccording to claim 1 wherein the first nucleotide sequence encodes acytokine.
 9. The method according to claim 8 wherein the cytokine isinterleukin 7 (IL-7).
 10. The method according to claim 9 wherein theIL-7 is human IL-7.
 11. The method according to claim 1 wherein theantigen receptor is a T cell receptor (TCR) wherein the TCR bindsspecifically to an MHC displaying a peptide fragment of a tumor antigenexpressed by cancer cells in the patient. 12-14. (canceled)
 15. Themethod according to claim 11 wherein the TCR comprises the amino acidsequence of any one of SEQ ID NOs: 5, 6 or
 11. 16. The method accordingto claim 11 wherein the antigen receptor is a chimeric antigen receptor(CAR) that binds specifically to a tumor antigen expressed by cancercells in the patient. 17-18. (canceled)
 19. The method according toclaim 11 wherein the tumour antigen is NY-ESO-1 or MAGE-A10. 20.(canceled)
 21. The method according to claim 1 wherein the nucleic acidconstruct is provided in a vector.
 22. The method according to claim 21wherein the vector is a lentiviral vector. 23-38. (canceled)
 39. Themethod according to claim 1 wherein the cancer is characterized by thepresence of one or more cancer cells which bind to said antigenreceptor.
 40. The method according to claim 1 wherein the modified Tcells are autologous to the patient.
 41. The method according to claim 1wherein the modified T cells are allogeneic to the patient. 42-49.(canceled)
 50. The method according to claim 16 wherein the tumorantigen is NY-ESO-1 or MAGE-A10.